Angiosarcoma is a biologically aggressive vascular malignancy with a high metastatic potential and subsequent mortality(1). At the time of diagnosis of angiosarcoma, 10[unreadable] 25% of patients already have metastatic disease. The 2- and 5-year overall survival for all patients with angiosarcoma is 50 and 30%, respectively, with a median overall survival of 24 months. For unresectable angiosarcoma, doxorubicin based regimens yield progression-free survival of 5 months, and paclitaxel achieves a progression-free survival of 7 months(2). Therefore there is a desperate need for novel therapies to improve survival in patients with this highly lethal disease. Our laboratory has recently discovered a novel signaling pathway responsible for angiosarcoma growth. While conducting genomic profiling of breast tumor vascular cells compared to normal vessels obtained by laser capture microdissection, we identified secreted frizzled related protein 2 (SFRP2) as a gene with increased expression in tumor endothelium. SFRP2 is a 33kd secreted protein involved in Wnt signaling. Our preliminary data shows that SFRP2 protein is present in 9/9 human angiosarcomas by IHC, and stimulates angiogenesis on the chick chorioallantoic membrane, induces endothelial cell migration and tube formation, and protects against hypoxia induced apoptosis. Silencing of SFRP2 in SVR angiosarcoma cells resulted in inhibition of tube formation, and stimulation of endothelial cells with SFRP2 resulted in an increase in nuclear NFATc3. NFAT is a transcription factor that plays a critical role in mediating angiogenic responses(3;4). NFAT nuclear localization is dependent on a dynamic import-export balance between the activity of the Ca2+/calmodulin-dependent phosphatase, calcineurin, and the activity of serine/threonine kinases(20). NFAT cannot normally enter the nucleus until it is dephosphorylated, but can be activated by calcineurin. Activated calcineurin dephosphorylates NFAT, which then translocates from the cytoplasm to the nucleus and results in transcription of genes involved in cell growth, differentiation, and cell cycle progression. Tacrolimus (FK506) is an immunosuppressive drug that binds to the immunophlin FKBP12 in lymphocytes. The FK506-FKBP12 complex associates with calcineurin and inhibits its phosphatase activity, which inhibits nuclear translocation of NFAT(5). Our preliminary data shows that tacrolimus inhibits angiosarcoma tube formation in vitro, and systemic administration inhibits the growth of the SVR angiosarcoma xenograft in nude mice by 46% at 20 days. Taken together, our data leads us to propose the following hypothesis: Blockade of SFRP2 will decrease angiogenesis and angiosarcoma growth and will synergistically improve tumor regression in combination with both chemotherapy and antiangiogenic therapy. The objective of this study is to show the efficacy of these drugs in angiosarcoma alone, and in combination with chemotherapy and antiangiogenic therapy, and to elucidate their molecular mechanism in inhibiting tumor growth. Successful completion of the project will lead to clinical trials of these agents in patients with angiosarcoma in future studies. PUBLIC HEALTH RELEVANCE: Angiogenesis is the growth of new capillary blood vessels, and is a critical component of solid tumor growth. We have discovered a novel protein, secreted-frizzled related protein 2, that is overexpressed in human angiosarcoma that is a potent stimulator of angiogenesis. The objective of this proposal is to characterize the molecular and cellular mechanism of this protein on angiosarcoma biology, and evaluate the efficacy of blockade of this signaling pathway on angiosarcoma xenograft growth.